MicroRNAs (miRNAs) are ~22 nucleotide (nt) ubiquitous gene regulators that modulate diverse cellular pathways including differentiation, proliferation and apoptosis, all critical to human development and disease. Accumulating evidence suggests that various miRNAs are aberrantly expressed in cancer cells, underscoring the importance of elucidating miRNA biogenesis mechanisms. In Herpesvirus saimiri (HVS) infected marmoset T cells, viral miRNAs are co-transcribed immediately downstream of viral small nuclear RNAs (snRNAs). These were the first snRNA-miRNA chimeras to be identified. They are cleaved apart by the host Integrator complex, bypassing canonical Microprocessor cleavage. The resulting miRNAs may contribute to T-cell leukemias and lymphomas, diseases caused by HVS infection. My search for snRNA-miRNA chimeras in mammals led to the unexpected discovery of m7G-capped precursor (pre-)miRNAs, which are alternatively exported by Exportin-1 (rather than the canonical Exportin-5) and generate only a single mature miRNA. MiR-320 is derived from a m7G-capped precursor and suppresses epithelial mammary tumors. The objectives of this proposal are to further delineate the two noncanonical miRNA biogenesis pathways (Aim 1), expand the repertoire of alternatively-processed miRNAs and identify their functions (Aim 2). In the K99 phase, Integrator complex will be affinity purified from nuclear extract using in vitro transcribed HVS miRNA substrate and recombinant Integrator will be assayed for activity (Aim 1a). An in vitro RNA polymerase II transcription system will be developed to assess potential transcription termination at the 32 end of m7G- capped pre-miRNAs (Aim 1b). For the projects in R00 phase, an siRNA/GFP system is being devised to screen for genes that affect miRNA biogenesis (Aim 1c). MiRNAs using the HVS miRNA or m7G-capped pre- miRNA biogenesis pathways will be sought in different species through in silico and knockdown studies (Aim 2a). To identify mRNA targets of the alternatively-processed miRNAs, I propose a method involving Argonaute- UV crosslinking and immunoprecipitation (CLIP) followed by intermolecular ligation between target mRNAs and viral miRNAs, which are paired within Argonaute (Aim 2b). Identified targets for HVS miRNAs and m7G-capped precursor-derived miRNAs will shed light on mechanisms underlying T-cell leukemias/lymphomas and other cancers. Deciphering the details of these two novel miRNA biogenesis pathways, which are distinct from the pathway that generates most cellular miRNAs, will potentiate promising therapeutics designed to modulate specific miRNAs to combat cancer and other malignancies induced by related human Herpesviruses.